1. Field of the Invention
This invention relates to a process for regenerating, in the presence of a lysate of a microorganism of the species Clostridium kluyverl, a number of coenzymes, such as adenosine triphosphate (ATP), acetyl coenzyme A (acetyl-CoA), acetyl phosphate (acetyl-P), oxidized or reduced nicotinamide adenine dinucleotide (NAD or NADH) and oxidized or reduced nicotinamide adenine dinucleotide phosphate (NADP or NADPH), in connection with a biochemical reaction in which the coenzymes are consumed. In this process another agent, either an alcohol or aldehyde, such as ethanol or acetaldehyde also is consumed.
2. Description of Related Art
Due to their ability to function at mild reaction conditions and their high selectivity, enzymes are becoming increasingly more important in chemical engineering applications. However, only relatively few of the enzymes described in the literature have achieved widespread use in industrial processes. Moreover, in only a limited number of examples have enzymes been used which are found only in the interior of cells. Among these latter enzymes are a group of cosubstrate-dependent enzymes which to date, have only been used in scientific investigations. Cosubstrate-dependent enzymes are enzymes which require the presence of certain other substrates in order to catalyze a chemical reaction (the "certain other substrates" are generally referred to as cofactors, coenzymes or cosubstrates). Certain coenzymes are consumed in stolchlometric quantities along with the substrate during the course of the reaction. Examples of these coenzymes are ATP, acetyl-CoA, acetyl-P, NAD(P) and NAD(P)H. Throughout this application, NAD and NADP will be referred to jointly as NAD(P), while their reduced forms, i.e., NADH and NADPH, similarly will be referred to jointly as NAD(P)H.
The significant expense associated with producing large quantities of these coenzymes has effectively precluded their use in the chemical engineering industry. In other words, enzymatic processes employing enzymes that require stoichiometric amounts of a coenzyme for activation generally cannot complete economically with other available synthesis methods.
In order to remedy this situation, attempts have been made to develop procedures for regenerating individual coenzymes. For example, it has been shown that NAD(P) can be converted into NAD(P)H inter alia by allowing the flavoenzymes lipoamide dehydrogenase (EC 1.6.4.3) or ferredoxin reductase (EC 1.6.99.4), or the extracts of certain microorganisms to act on NAD(P) in the presence of reduced methyl viologen (1,1'-dimethyl-4,4-bipyridinium). The reduced methyl viologen can be regenerated by contacting with hydrogen in the presence of hydrogenase or by direct current (See C.-H. Wong et al. J. Am. Chem. Soc. 103 (1981), pages 6227-6228 and J. Bader et al. J. Biotechn. 1 (1984), pages 95-109).
While these processes significantly reduce the quantity of NAD(P)H needed for an enzymatic reaction, the Wong et al. process is disadvantaged, for example, by its reliance on hydrogen as a reactant and the Bader et al. process is complicated, for example, by the need to employ an electrochemical cell. Similar reactions also have been proposed for regenerating other coenzymes, such as, for example, regenerating acetyl-CoA, acetylphosphate or ATP. Unfortunately, the processes require enzymes isolated and purified by complicated methods and/or these processes require the presence of other substrates which themselves are difficult to obtain (G. M. Whitesides, Chi H. Wong, Aldrichimica Acta 16 (1983) 27-34).
Accordingly, a need exists in the art of enzyme catalyzed reactions for a simple process for regenerating coenzymes individually or simultaneously in mixtures. An object of the present invention therefore is to provide such a regeneration process which employs alkanols and alkanals containing from 2 to 4 carbon atoms and an enzyme preparation easily obtained by lysis of a cell suspension.